The goal of this proposal is to develop a system for chronic in vivo imaging of neuronal morphology in the intact rodent visual cortex. A system that can be used to directly test predictions regarding the site and extent of structural dynamics that occur in visual cortex on a day to day basis and in response to visual input. By combining this innovative imaging technology with state of the art mouse genetics we can begin to address the molecular basis of cortical structural plasticity. This integrative approach will not only revolutionize our ability to understand this fundamental aspect of brain function, but can identify molecules with therapeutic potential that can promote plasticity in visual cortex and may be used to compensate for insults at lower levels of the visual pathway. To monitor in vivo structural dynamics of neurons in the mammalian visual cortex, we will fabricate a custom designed two-photon microscope for imaging single neurons within a three-dimensional volume of the mouse visual cortex with high fidelity and at short time scales. Multi-photon microscopy allows for vital imaging deep into scattering tissue at sub-cellular resolution with minimal photodamage or phototoxicity. We will use this microscope to image in vivo and reconstruct entire dendritic trees and axon collaterals of EGFP (enhanced green fluorescent protein) expressing neurons in the visual cortex of transgenic mice. These mice express EGFP driven by the thy1 promoter in a random subset of cells sparsely distributed within the superficial cortical layers. The EGFP fills the cells so that they are brightly fluorescent all the way to the tips of their terminals, allowing visualization of their entire structure including filopodia and dendritic spines. We will image these neurons over time in anesthetized animals through a cranial window that allows repeated access to the same cells. To identify molecules that play a role in the structural remodeling that underlies visual cortical plasticity, we will cross mouse lines that are knockouts or transgenic for candidate plasticity genes (CPGs) with the thyl-EGFP transgenic line. This will create mice that are deficient or overexpressing a specific gene and have fluorescent cells in their cortex. Monitoring the structural dynamics of cortical neurons in these mice and comparing them to those of EGFP labeled cortical neurons from wild-type animals, will reveal to what extent the manipulated genes play a role in structural plasticity of the mammalian cortex. Genes could also be introduced into the cortex transiently by virally mediated gene transfer. Some of the first genes tested will be CPGs that we isolated in a screen for activity-regulated genes involved in synaptic plasticity.